key: cord-0275247-as51onhz
authors: Ponti, András K.; Zangara, Megan T.; O’Connor, Christine M.; Johnson, Erin E.; McDonald, Christine
title: N-phosphonacetyl-L-aspartate enhances type I interferon anti-viral responses through activation of non-canonical NOD2 signaling
date: 2022-02-08
journal: bioRxiv
DOI: 10.1101/2022.02.08.479597
sha: 3f0128f64da4fd52509c09e8f24df6b126a52a91
doc_id: 275247
cord_uid: as51onhz

Type I interferon production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens. Dysregulation of this response can result in uncontrolled infection, inflammation, and autoimmune disease. Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights into infection control strategies and autoimmune disease therapies. Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that acts as both a bacterial sensor protein and a mediator of antiviral responses. Antibacterial functions of NOD2 are enhanced by treatment with the small molecule inhibitor of pyrimidine biosynthesis N-phosphonacetyl-L-aspartate (PALA), though how this might function in the host antiviral response remains unknown. Therefore, we tested the ability of PALA to enhance NOD2-dependent antiviral responses. Alone, PALA treatment of macrophages was not sufficient to induce interferon β (IFNβ) production or ISG expression. Instead, PALA synergized with IFNβ stimulation to enhance expression and activation of interferon-stimulated gene factor 3 (ISGF3) and induce the upregulation of a subset of ISGs in co-treated cells. Furthermore, PALA treatment of epithelial cells resulted in impaired viral replication of the herpesvirus, human cytomegalovirus. Induction of the PALA-enhanced antiviral response required activation of non-canonical NOD2 signaling mediated by mitochondrial antiviral-signaling protein (MAVS) and interferon response factor 1 (IRF1), rather than the classical receptor-interacting serine/threonine protein kinase 2 (RIP2) pathway or other IRFs previously reported to mediate NOD2 antiviral responses. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses and antiviral activity. Significance Statement Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights to improve infection control strategies and autoimmune disease therapies. This work demonstrates that the pyrimidine synthesis inhibitor N-phosphonacetyl-L-aspartate synergizes with type I interferon to enhance antiviral responses through activation of a non-canonical NOD2 signaling pathway. These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses.

Type I interferon production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens. 

Understanding the molecular mechanisms shaping the strength of type I interferon signaling may provide critical insights to improve infection control strategies and autoimmune disease therapies. This work demonstrates that the pyrimidine synthesis inhibitor N-phosphonacetyl-L-aspartate synergizes with type I interferon to enhance antiviral responses through activation of a non-canonical NOD2 signaling pathway.

These findings highlight pyrimidine metabolism enzymes as controllers of antimicrobial responses and suggest novel mechanisms for the modulation of type I interferon responses.

Type I interferon (IFN-I) production and the expression of interferon-stimulated genes (ISGs) are key components of an innate immune response to many microbial pathogens (1) . The interferon family is comprised of 14 IFNα subtypes, IFNβ, IFNω, IFNε, IFNĸ, IFNζ, and IFNτ, and is widely recognized to induce both an antiviral response within The canonical STAT complex activated by IFN-I is interferon stimulated gene factor 3 (ISGF3) that is comprised of STAT1, STAT2, and IRF9. The binding of ISGF3 to specific DNA sequences called interferon stimulated response elements (ISREs) activates the transcription of a broad array of ISGs to induce an antiviral state, upregulate immunomodulatory molecules, as well as further amplify IFN-I signaling.

One PRR known to induce IFNβ production is nucleotide-binding oligomerization domain 2 (NOD2) (5) . NOD2 is best known for its role as a cytoplasmic bacterial sensor protein, but also mediates antiviral responses to ssRNA and dsDNA viruses, such as respiratory syncytial virus (RSV) (6) and human cytomegalovirus (HCMV) (7, 8) .

Classically, NOD2 senses the peptidoglycan fragment muramyl dipeptide (MDP) component of the bacterial cell wall and triggers a signaling cascade initiated by receptor-interacting serine/threonine protein kinase 2 (RIP2), resulting in the activation NFĸB, AP-1, and IRF5 transcription factors. Activation of IRF5 by NOD2 is essential for the induction of Ifnβ transcription in response to Mycobacterium tuberculosis infection (9) . IFNβ production can also be stimulated as part of an antiviral response to RSV infection via the formation of a protein complex containing NOD2 and mitochondrial antiviral-signaling protein (MAVS) that results in the activation of IRF3 (6) .

Our prior work demonstrated that NOD2 interacts with the pyrimidine synthesis enzyme carbamoyl-phosphate synthetase 2/ aspartate transcarbamylase/ dihydroorotase (CAD) and that CAD negatively regulates NOD2 antibacterial activity (10) . Conversely, NOD2 antibacterial activity is enhanced by the CAD inhibitor Nphosphonacetyl-L-aspartate (PALA) (10, 11) . Interestingly, other pyrimidine synthesis inhibitors have been developed to target this metabolic pathway, and many stimulate broad-spectrum antiviral activity in addition to suppressing nucleotide synthesis (12) .

Therefore, we investigated whether PALA modulates IFN-I production through activation of NOD2 antiviral signaling in macrophages. Similar to other pyrimidine synthesis inhibitors, we observed that PALA amplified the production of IFNβ in macrophages treated with IFN-I. However, unlike inhibitors targeting other pyrimidine synthesis pathway enzymes, the PALA-enhanced IFN-I response resulted in upregulated expression and prolonged activation of ISGF3. The PALA/IFN-I amplified response required activation of non-canonical NOD2 signaling mediated by MAVS and IRF1, rather than the classical RIP2 pathway or other IRFs previously reported to mediate NOD2 antiviral responses. These findings uncover a novel signaling pathway that modulates IFN-I responses.

Inhibition of enzymes governing pyrimidine synthesis enhances the type I interferon response via distinct mechanisms. De novo pyrimidine synthesis is a multistep process requiring six different enzymatic activities housed in three proteins to generate pyrimidines (13) (SI Appendix, Fig. S1 ). Pharmacological inhibitors have been developed to target each enzyme of this metabolic pathway and many of these drugs also stimulate broad-spectrum antiviral immune responses; however, it is unclear whether these compounds induce an antiviral state using the same mechanism of action (12) . Fig. S2 ). PALA stimulation alone was not sufficient to increase either the transcript or protein levels of ISGF3 components (Fig. 2) , suggesting that PALA synergizes with IFNβ to upregulate ISGF3 transcription.

We also found that PALA co-treatment with IFN-I correlated with increased ISGF3 expression and activation. Our data reveal that PALA co-treatment increased of IE genes and their translated proteins induce expression of early viral genes, whose proteins are required for viral DNA replication (18, 19) . PALA treatment resulted in higher accumulation of IE1 at all time points, but markedly reduced levels of pUL44 at 72h and 96h post-infection (Fig. 3D ). This indicates that PALA limits translation of early proteins, resulting in repression of viral replication independent of cellular pyrimidine depletion. Collectively, these data demonstrate that PALA, under conditions that stimulate an IFN-I response, effectively induces an antiviral state.

CAD binds to and negatively regulates the antibacterial activity of the intracellular pattern recognition receptor, NOD2 (10), thus demonstrating a direct link between pyrimidine synthesis and innate immune modulation. We tested whether the enhancement of IFN-I responses by PALA co-treatment was dependent on NOD2 expression. Bone marrowderived macrophages (BMDMs) were isolated from C57BL/6 (WT) or Nod2 -/-(NOD2KO) mice and stimulated with IFNβ, PALA, or a combination of IFNβ and PALA for 24h and STAT1 expression levels were determined by immunoblot. Although IFNβ stimulated STAT1 protein expression in both WT and NOD2KO macrophages, NOD2KO macrophages were unresponsive to PALA treatment (Fig. 4A ). These results indicate that NOD2 expression is required to mediate PALA enhancement of a type I IFN response.

NOD2 forms a protein-protein complex with CAD (10) and knockdown of CAD expression or PALA treatment enhances NOD2 activity (10, 11) . Therefore, we next investigated whether PALA relieves CAD inhibition of NOD2 activity through disruption of the CAD-NOD2 protein complex. PALA treatment of HEK293T cells expressing epitopetagged CAD and NOD2 proteins decreased the amount of CAD co-immunoprecipitating with NOD2 (Fig. 4B ). We also quantitatively assessed the PALA-induced disruption of the CAD-NOD2 complex using the Lumier with Bacon assay (20) that measures the amount of a Renilla-tagged NOD2 associating with a quantified amount of plate-bound Flag-CAD through sequential luminescent and ELISA readings of the same well. As in PALA induces activation of a non-canonical NOD2 signaling pathway mediated by IRF1.

It is accepted that IRFs signal downstream of MAVS as part of the IFN-I response to viruses (22) . For example, the NOD2-MAVS-IRF3 axis mediates an IFN-I response to ssRNA and respiratory syncytial virus (RSV) infection (6) . Surprisingly, PALA did not 

Administration of recombinant IFN-I is used clinically to control viral infections, such as hepatitis C, hepatitis B, and human immunodeficiency virus, or as antineoplastic therapies for hairy cell leukemia, melanoma, and breast cancer (23) . Although IFN-I can be used as a single agent, is it is often administered with another drug, such as the nucleotide synthesis inhibitors ribavirin or gemcitabine, to increase therapeutic efficacy. Our data indicate that PALA treatment interferes with HCMV infection by suppressing expression of viral early protein synthesis in epithelial cells, suggesting that this CAD inhibitor has immune modulatory antiviral actions in addition to suppression of cellular pyrimidine synthesis. HCMV infection results in increased pyrimidine synthesis flux to provide nucleotides for genome replication and UDP-sugars for glycosylation of viral envelope proteins (26) . Inhibition of pyrimidine synthesis by PALA or DHODH inhibitors successfully blocked HCMV replication and production of glycosylated envelope proteins required for infectious virions (26) . However, in contrast to our findings that PALA caused a reduction in pUL44 expression, the prior study of HCMV infected embryonic lung fibroblasts showed that accumulation of viral proteins were unaffected by PALA treatment, although viral DNA abundance was substantially reduced and not rescued by addition of exogenous uridine (26) . We postulate the difference between our findings and the previous work may be the higher level of basal NOD2 expression in epithelial cells versus fibroblasts. In support of this, others have shown NOD2 expression is undetectable in human foreskin fibroblasts, though HCMV infection robustly upregulates NOD2 expression in these cells (27) . This also raises the possibility of tissue-specific differences in the antiviral response, as fibroblasts of different tissue origin may differentially regulate NOD2 (26, 27) . Similar to our results, exogenous NOD2 expression prior to HCMV infection resulted in reduced pUL44 expression and decreased viral replication (27) . These findings suggest that through NOD2 stimulation, PALA inhibits HCMV replication independent of mechanisms involving pyrimidine depletion.

Multiple nucleotide synthesis inhibitors link nucleotide biosynthesis with regulation of innate immune responses that potentiate an anti-viral response through upregulation of ISGs; however, the molecular mechanisms underlying these responses are not well-defined (12) . Similar to the actions of PALA, the DHODH inhibitors, DD78

and brequinar, synergize with ssRNA stimulation to enhance production of ISGs in an IRF1-dependent manner (16) . However, our data indicate the signaling pathways that MAVS expression is required for upregulation of IFNβ-stimulated STAT1 expression by PALA. Raw264.7 cells were transfected with non-targeting (Control) or MAVS-targeting RNAi for 72h. Cells were treated as in A. and analyzed for STAT1, MAVS, and tubulin protein levels (n=4). C. B16-Blue IFNα/β reporter cells were transfected with non-targeting (Control) or MAVS-targeting RNAi for 72h. Cells were treated as in A. and ISRE reporter assayed through quantification of SEAP levels in the media by colormetric assay (n=4 performed in triplicate). Data presented as mean±SEM; significance determined by one-way ANOVA and Tukey's multiple comparisons test; ns=not significant, * p≤0.05 vs. IFN. Inset shows MAVS and tubulin protein levels to confirm RNAi-mediated knockdown of expression. , or costimulation with IFNβ and PALA for 1h. Cell lysates were fractionated into cytoplasmic and nuclear fractions and immunoblots performed to visualize the localization of IRF1, IRF3, and IRF5 (n=3; representative experiment shown). Blots were also probed for the nuclear protein TBP and the cytosolic protein tubulin as controls. IRF1 nuclear protein levels were quantified by densitometry and fold change relative to NT (left panel). B. Enhancement of IFNβ activation of IRF1 by PALA can be visualized by immunofluorescent confocal microscopy. Raw264.7 cells were treated as in A., co-stained with anti-IRF1 antibody (red) and DAPI (blue), and visualized by fluorescent confocal microscopy. Intensity of IRF1 nuclear signal was quantitated using Image-Pro software. Measurements represent 5 replicate fields from 4 individual experiments. Data presented as mean±SEM; significance determined by one-way ANOVA and Tukey's multiple comparisons test; ns=not significant, **** p≤0.0001 vs. NT, ++++ p≤0.0001 vs. IFN. C. IRF1 expression is required for increased IFNβ-stimulated ISRE transcription in response to PALA treatment. B16-Blue IFNα/β reporter cells were transfected with non-targeting (Control) or IRF1targeting RNAi for 72h. Cells were treated as in A. for 24h and ISRE reporter assayed through quantification of SEAP levels in the media by colormetric assay (n=4 performed in triplicate). Data presented as mean±SEM; significance determined by one-way ANOVA and Tukey's multiple comparisons test; ns=not significant, ++++p≤0.0001 vs. IFN. Inset shows immunoblot of IRF1 and tubulin protein levels to show RNAi-mediated knockdown of expression. D. Activation of IRF1 by PALA is NOD2-dependent. BMDM from C57BL/6 (WT) or Nod2 -/-(NOD2KO) mice were stimulated and analyzed for IRF1 localization by immunofluorescent confocal microscopy as in B. 

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Activation of nucleotide oligomerization domain 2 (NOD2) by human cytomegalovirus initiates innate immune responses and restricts virus replication

We would like to thank Dr. George Stark for the generous gift of cell lines, Dr. Derek Abbott for providing us with bone marrow from RIP2KO mice, Dr. Judy Drazba and Dr.Gauravi Deshpande in the Cleveland Clinic Imaging Core for assistance with confocal